DESCRIPTION: The regeneration of damaged or diseased skeletal tissues remains a significant clinical challenge and cause for human disability and discomfort. Tissue engineering and regenerative medicine are emerging as promising strategies for treating these conditions by creating living tissue substitutes composed of cells, bioactive factors, and a biodegradable scaffold. Tissue engineering has been particularly successful in creating uniform tissues, such as skin and cartilage. However, many injuries and diseases affect the interfaces between tissues, such as the transition between bone and cartilage. Many regenerative medicine studies that address these more complex tissues have been successful in engineering tissues in vitro with combinations of progenitor cells and biomaterials. However, these approaches typically require elaborate, laborious, and expensive procedures for cell isolation, expansion, and in vitro cell conditioning for proper cell differentiation and tissue formation. A primary challenge has been the translation of these promising preclinical studies into methods that are not only straightforward to practice but also effectively direct cell differentiation and tissue formation tat recapitulates the complexity of natural tissues. Technologies that capitalize on advances in cell engineering but minimize in vitro cell manipulation can greatly enhance the promise of regenerative medicine. This project develops a method to deliver bioactive factors to cells in a precise spatial pattern within a three dimensional scaffold. As a result, stem cells seeded onto these scaffolds or endogenous progenitor cells that infiltrate the scaffold will be stimulated to produce different tissue types in predefined patterns. In this study, we will first generate osteochondral tissues with human mesenchymal stem cells through spatially controlled gene transfer of differentiation factors. Precise spatial control will be enabled by innovative methods of biomaterial-mediated gene delivery and a microscale weaving technique for creating 3D polymer scaffolds. We will then expand this in vitro approach to a rabbit model of microfracture surgery, where the scaffold is populated by endogenous progenitor cells from the bone marrow that are instructed to form tissues in situ based on viral transgene delivery from the scaffold. This work is significant to developing tissue engineering strategies for creating complex structures that recapitulate the heterogeneity and function of native tissue interfaces and minimize in vitro cell manipulations.